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Tobacco (Nicotiana tabacum) is cultivated and consumed worldwide. It requires great amounts of nitrogen (N) to achieve the best yield and quality. With a view to sustainable and environmentally friendly agriculture, developing new genotypes with high productivity under low N conditions is an important approach. It is unclear how genes in tobacco are expressed at the cellular level and the precise mechanisms by which cells respond to environmental stress, especially in the case of low N. Here, we characterized the transcriptomes in tobacco leaves grown in normal and low‐N conditions by performing scRNA‐seq. We identified 10 cell types with 17 transcriptionally distinct cell clusters with the assistance of marker genes and constructed the first single‐cell atlas of tobacco leaves. Distinct gene expression patterns of cell clusters were observed under low‐N conditions, and the mesophyll cells were the most important responsive cell type and displayed heterogene responses among its three subtypes. Pseudo‐time trajectory analysis revealed low‐N stress decelerates the differentiation towards mesophyll cells. In combination with scRNA‐seq, WGCNA, and bulk RNA‐seq results, we found that genes involved in porphyrin metabolism, nitrogen metabolism, carbon fixation, photosynthesis, and photosynthesis‐antenna pathway play an essential role in response to low N. Moreover, we identified COL16, GATA24, MYB73, and GLK1 as key TFs in the regulation of N‐responsive genes. Collectively, our findings are the first observation of the cellular and molecular responses of tobacco leaves under low N stress and lay the cornerstone for future tobacco scRNA‐seq investigations. [ABSTRACT FROM AUTHOR]

Pre‐existing drug resistance and tumorigenicity of cancer cells are highly correlated with therapeutic failure and tumor growth. However, current cancer models are limited in their application to the study of intratumor functional heterogeneity in personalized oncology. Here, an innovative two‐dimensional (2D) and three‐dimensional (3D) model for patient‐derived cancer cells (PDCCs) and air–liquid interface (ALI) organotypic culture is established from colorectal cancer (CRC). The PDCCs recapitulate the genomic landscape of their parental tumors with high efficiency, high proliferation rate, and long‐term stability, while corresponding ALI organotypic cultures retain histological architecture of their original tumors. Interestingly, both 2D and 3D models maintain the transcriptomic profile of the corresponding primary tumors and display the same trend in response to 5‐Fluoruracil, regardless of their difference in gene expression profiles. Furthermore, single‐cell‐derived clones() are efficiently established and pre‐existing drug‐resistant clones and highly tumorigenic clones within individual CRC tumors are identified. It is found that tumorigenic cancer cells do not necessarily possess the stem cells characteristics in gene expression. This study provides valuable platform and resource for exploring the molecular mechanisms underlying the pre‐existing drug resistance and tumorigenicity in cancer cells, as well as for developing therapeutic targets specifically for pre‐existing drug‐resistant or highly tumorigenic clones. [ABSTRACT FROM AUTHOR]

Pre‐existing drug resistance and tumorigenicity of cancer cells are highly correlated with therapeutic failure and tumor growth. However, current cancer models are limited in their application to the study of intratumor functional heterogeneity in personalized oncology. Here, an innovative two‐dimensional (2D) and three‐dimensional (3D) model for patient‐derived cancer cells (PDCCs) and air–liquid interface (ALI) organotypic culture is established from colorectal cancer (CRC). The PDCCs recapitulate the genomic landscape of their parental tumors with high efficiency, high proliferation rate, and long‐term stability, while corresponding ALI organotypic cultures retain histological architecture of their original tumors. Interestingly, both 2D and 3D models maintain the transcriptomic profile of the corresponding primary tumors and display the same trend in response to 5‐Fluoruracil, regardless of their difference in gene expression profiles. Furthermore, single‐cell‐derived clones() are efficiently established and pre‐existing drug‐resistant clones and highly tumorigenic clones within individual CRC tumors are identified. It is found that tumorigenic cancer cells do not necessarily possess the stem cells characteristics in gene expression. This study provides valuable platform and resource for exploring the molecular mechanisms underlying the pre‐existing drug resistance and tumorigenicity in cancer cells, as well as for developing therapeutic targets specifically for pre‐existing drug‐resistant or highly tumorigenic clones. [ABSTRACT FROM AUTHOR]

To determine the optimal activity region of the dopachrome tautomerase gene (DCT) promoter in sheep skin melanocytes, five eukaryotic expression vectors were constructed containing DCT promoter fragments of different lengths (441 bp, 652 bp, 834 bp, 1155 bp, and 1504 bp) ligated to the pLV.ExSi.P/Puro-mouse DCT-eGFP vector. These constructs were transiently transfected into sheep skin melanocytes using liposome transfection technology. GFP (Green Fluorescent Protein) expression varied among the groups transfected with different constructs as follows: 652 bp > 834 bp > 441 bp > 1504 bp > 1155 bp. GFP expression was significantly higher in the 652 bp and 834 bp groups than in the 1155 bp group (p < 0.01), while expression in the 652 bp group was significantly higher than in the 1504 bp group (p < 0.05). Additionally, we identified elements participating in the initiation of gene transcription (a CAAT box), cell type-specific expression (M-box), and the regulation of gene expression (E-box) in the sheep DCT promoter. In conclusion, the optimal active region of the sheep DCT promoter is located between -593 bp and + 41 bp. [ABSTRACT FROM AUTHOR]

The SCN5A gene encodes cardiac sodium channel Na v 1.5 and causes lethal ventricular arrhythmias/sudden death and atrial fibrillation (AF) when mutated. MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression, and involved in the pathogenesis of many diseases. However, little is known about the regulation of SCN5A by miRNAs. Here we reveal a novel post-transcriptional regulatory mechanism for expression and function of SCN5A /Na v 1.5 via miR-192-5p . Bioinformatic analysis revealed that the 3′-UTR of human and rhesus SCN5A , but not elephant, pig, rabbit, mouse, and rat SCN5A , contained a target binding site for miR-192-5p and dual luciferase reporter assays showed that the site was critical for down-regulation of human SCN5A . With Western blot assays and electrophysiological studies, we demonstrated that miR-192-5p significantly reduced expression of SCN5A and Na v 1.5 as well as peak sodium current density I Na generated by Na v 1.5. Notably, in situ hybridization, immunohistochemistry and real-time qPCR analyses showed that miR-192-5p was up-regulated in tissue samples from AF patients, which was associated with down-regulation of SCN5A /Na v 1.5. These results demonstrate an important post-transcriptional role of miR-192-5p in post-transcriptional regulation of Na v 1.5, reveal a novel role of miR-192-5p in cardiac physiology and disease, and provide a new target for novel miRNA-based antiarrhythmic therapy for diseases with reduced I Na . [ABSTRACT FROM AUTHOR]

In the present study we investigated the developmental expression patterns of Lbx1 gene in skeletal muscle of different genetic profile pigs after birth. A total of 28 Mashen pigs and 28 Large White pigs from seven development stages of 1, 30, 60, 90, 120, 150, 180 d were used and the expression patterns in longissimus dorsi were studied by quantitative real time-polymerase chain reaction and western blot in this study. The results showed that the breed, age, and the interaction of breed and age significantly affected the Lbx1 mRNA and protein expressions in longissimus dorsi (P<0.05). In Large White, the amount of Lbx1 protein was increased from new birth to 30 d, and decreased after 30 d to the lowest level at 180 d. In Mashen pig, Lbx1 protein level showed an increase trend with the age increase before 90 d, then decreased to the lowest level at 120 d, and then gradually increased upwards to the expression peak at 180 d. Before 120 d, the Lbx1 protein content in Large White was higher than that in Mashen pig, except for at 60 d. Afterwards, the protein level in Mashen was higher than that in Large White. The Lbx1 expression pattern was related to the age and genetic profiles of Mashen and Large White pigs, which indicates that Lbx1 could participate in the muscle development through activating the satellite cells of skeletal muscle after birth. [ABSTRACT FROM AUTHOR]

Background: Intestinal metaplasia (IM) of gastric epithelium has traditionally been regarded as an irreversible stage in the process of the Correa cascade. Exploring the potential molecular mechanism of IM is significant for effective gastric cancer prevention. Methods: The GSE78523 dataset, obtained from the Gene Expression Omnibus (GEO) database, was analyzed using RStudio software to identify the differently expressed genes (DEGs) between IM tissues and normal gastric epithelial tissues. Subsequently, gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, Gene Set Enrichment Analysis (GESA), and protein-protein interaction (PPI) analysis were used to find potential genes. Additionally, the screened genes were analyzed for clinical, immunological, and genetic correlation aspects using single gene clinical correlation analysis (UALCAN), Tumor–Immune System Interactions Database (TISIDB), and validated through western blot experiments. Results: Enrichment analysis showed that the lipid metabolic pathway was significantly associated with IM tissues and the apolipoprotein B (APOB) gene was identified in the subsequent analysis. Experiment results and correlation analysis showed that the expression of APOB was higher in IM tissues than in normal tissues. This elevated expression of APOB was also found to be associated with the expression levels of hepatocyte nuclear factor 4A (HNF4A) gene. HNF4A was also found to be associated with immune cell infiltration to gastric cancer and was linked to the prognosis of gastric cancer patients. Moreover, HNF4A was also highly expressed in both IM tissues and gastric cancer cells. Conclusion: Our findings indicate that HNF4A regulates the microenvironment of lipid metabolism in IM tissues by targeting APOB. Higher expression of HNF4A tends to lead to a worse prognosis in gastric cancer patients implying it may serve as a predictive indicator for the progression from IM to gastric cancer. [ABSTRACT FROM AUTHOR]

BACKGROUND AND OBJECTIVES: MicroRNA (miR-221/222) is frequently overexpressed in many cancers and is associated with poor prognosis. However, the role of miR-221/222 in retinoblastoma (RB) remains unclear. This study aimed to detect the clinical significance of miR-221/222 in RB patients and explore its role in RB cells in vitro. METHODS: Expression of miR-221/222 was assessed in fresh RB tissue collected from 64 eyes and normal retinal tissues from 18 unrelated donor cadaver eyes by quantitative real time RT-PCR analysis (qRT-PCR), and correlated with the histopathological findings. Human RB Y79 cells were transfected with miR-221/222 precursors or inhibitors to overexpress or downregulate miR-221/222 expression, respectively, using Lipofectamine 2000 reagent. The biological effects of miR-221/222 were then assessed by cell viability assays, colony formation assays, apoptosis detection assays, Matrigel ® invasion assays, and wound-healing assays. RESULTS: Higher miR-221/222 expression was detected in RB tissues compared to that of the normal retinal tissues ( p < 0.001). Higher miR-221/222 expression was correlated with invasion in patients with RB. Targeting of miR-221/222 induced apoptosis and inhibited Y79 cell proliferation, migration, and invasion in vitro. However, overexpression of miR-221/222 promoted Y79 cell proliferation, migration, and invasion in vitro. CONCLUSIONS: Overexpression of miR-221/222 was associated with tumor invasiveness in patients with RB. The miR-221/222 cluster might be used as a potential therapeutic strategy in clinical practice. [ABSTRACT FROM AUTHOR]

Clear cell renal cell carcinoma (ccRCC) is an immunogenic tumor, and investigating the immunorelated genes is essential. To investigate the immunoprognostic genes of ccRCC, we analyzed the data assimilated from a public database (The Cancer Genome Atlas (TCGA) database and the gene expression omnibus (GEO) database) using bioinformatics. Then, an immunoprognosis model was constructed to identify four hub genes with moderate predictive values for the prognosis of ccRCC patients. These four genes were associated with the prognosis of ccRCC patients based on Oncomine and Gena Expression Profiling Interactive Analysis (GEPIA) databases. The correlation analysis between the immune infiltrate, immune checkpoints, and immunotherapy and this immunoprognosis model showed that immune infiltration could predict the immunotherapy effects. We also conducted a quantitative real-time polymerase chain reaction analysis and found that the expressions of three hub genes were associated with tumor progression (P<0.1). In conclusion, four genes that may serve as potential biomarkers in ccRCC were identified with respect to prognosis. [ABSTRACT FROM AUTHOR]

Key message: Wheat MAPK member TaMPK4 responds to abiotic stresses of Pi and N deprivations and high salinity and is crucial in regulating plant tolerance to aforementioned stresses. Abstract: Mitogen-activated protein kinase (MAPK) cascades are important signal transduction modules in regulating plant responses to various environmental stresses. In this study, a wheat MAPK member referred to TaMPK4 was characterized for its roles in mediating plant tolerance to diverse stresses. TaMPK4 shares conserved domains generally identified in plant MAPKs and possesses in vitro kinase activity. Under stresses of Pi and N deprivations and high salinity, TaMPK4 was strongly upregulated and its expressions were restored upon recovery treatments from above stresses. Sense- and antisense-expressing TaMPK4 in tobacco significantly modified plant growth under the stress conditions and dramatically modified the root architecture through transcriptional regulation of the auxin transport-associated genes NtPIN3 and NtPIN9, whose downregulated expressions dramatically reduced the root growth. Compared with wild type (WT), the antioxidant enzymatic activities under the stress conditions, P accumulation under P deprivation, and N amount under N deficiency were altered dramatically in the transgenic plants, showing higher in the TaMPK4-overexpressing and lower in the TaMPK4-knockout plants, which were in concordance with the modified expressions of a set of antioxidant enzyme genes ( NtPOD2;1, NtPOD9, NtSOD2, NtFeSOD, and NtCAT), two phosphate transporter genes ( NtPT and NtPT2), and two nitrate transporter genes ( NtNRT1.1- s and NtNRT1.1- t), respectively. Downregulated expression of above genes in tobacco largely reduced the plant growth, and Pi and N acquisitions under the stress conditions. TaMPK4 also involved regulations of plant K and osmolyte contents under high salinity. Thus, TaMPK4 is functional in regulating plant tolerance to diverse stresses through modifying various biological processes. [ABSTRACT FROM AUTHOR]

Interplay of pioneer transcription factor forkhead box A1 (FOXA1) and estrogen receptor has been implicated in sexual dimorphism in hepatocellular carcinoma (HCC), but etiological relevance of its polymorphism was unknown. In the case control study (1152 patients versus1242 controls), we observed significant increase in HCC susceptibility in hepatitis B virus carriers associated with a non-synonymous Thr83Ala variant of FOXA1 (odds ratio [OR], 1.28; 95% confidence interval [CI], 1.11−1.48, for Ala83-containing genotype, after validation in an independent population with 933 patients versus 1030 controls), a tightly linked (CGC)5/6or7 repeat polymorphism at its promoter (OR 1.32; 95% CI 1.10–1.60, for (CGC)6or7-repeat-containing genotype), and their combined haplotype (OR 1.50; 95% CI 1.24–1.81, for (CGC)6or7−Ala83 haplotype). The susceptible FOXA1-Ala83 impairs its interaction with ERα, attenuates transactivation toward some of their dual target genes, such as type 1 iodothyronine deiodinase, UDP glucuronosyltransferase 2 family, polypeptide B17 and sodium/taurocholate cotransporting polypeptide, but correlates with strengthened cellular expression of α-fetoprotein (AFP) and elevated AFP serum concentration in HCC patients (n = 1096). The susceptible FOXA1 cis -variant with (CGC)6or7 repeat strengthens the binding to transcription factor early growth response 1 and enhances promoter activity and gene expression. Evolutionary population genetics analyses with public datasets reveal significant population differentiation and unique haplotype structure of the derived protective FOXA1-Thr83 and suggest that it may have undergone positive natural selection in Chinese population. These findings epidemiologically highlight the functional significance of FOXA1-ERα transcriptional program and regulatory network in liver cancer development. [ABSTRACT FROM AUTHOR]

Background The CAV1 gene encodes caveolin-1 expressed in cell types relevant to atherosclerosis. Cav-1 -null mice showed a protective effect on atherosclerosis under the ApoE − / − background. However, it is unknown whether CAV1 is linked to CAD and MI in humans. In this study we analyzed a tagSNP for CAV1 in intron 2, rs3807989, for potential association with CAD. Methods and Results We performed case–control association studies in three independent Chinese Han populations from GeneID, including 1249 CAD cases and 841 controls in Population I, 1260 cases and 833 controls in Population II and 790 cases and 1212 controls in Population III (a total of 3299 cases and 2886 controls). We identified significant association between rs3807989 and CAD in three independent populations and in the combined population ( P adj = 2.18 × 10 −5 , OR = 1.19 for minor allele A). We also detected significant association between rs3807989 and MI ( P adj = 5.43 × 10 −5 , OR = 1.23 for allele A). Allele A of SNP rs3807989 was also associated with a decreased level of LDL cholesterol. Although rs3807989 is a tagSNP for both CAV1 and nearby CAV2 , allele A of SNP rs3807989 was associated with an increased expression level of CAV1 (both mRNA and protein), but not CAV2 . Conclusions The data in this study demonstrated that rs3807989 at the CAV1 / CAV2 locus was associated with significant risk of CAD and MI by increasing expression of CAV1 (but not CAV2 ). Thus, CAV1 becomes a strong candidate susceptibility gene for CAD/MI in humans. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is the most common cardiac arrhythmia at the clinic. Recent GWAS identified several variants associated with AF, but they account for <10% of heritability. Gene-gene interaction is assumed to account for a significant portion of missing heritability. Among GWAS loci for AF, only three were replicated in the Chinese Han population, including SNP rs2106261 (G/A substitution) in ZFHX3, rs2200733 (C/T substitution) near PITX2c, and rs3807989 (A/G substitution) in CAV1. Thus, we analyzed the interaction among these three AF loci. We demonstrated significant interaction between rs2106261 and rs2200733 in three independent populations and combined population with 2,020 cases/5,315 controls. Compared to non-risk genotype GGCC, two-locus risk genotype AATT showed the highest odds ratio in three independent populations and the combined population (OR=5.36 (95% CI 3.87-7.43), P=8.00×10-24). The OR of 5.36 for AATT was significantly higher than the combined OR of 3.31 for both GGTT and AACC, suggesting a synergistic interaction between rs2106261 and rs2200733. Relative excess risk due to interaction (RERI) analysis also revealed significant interaction between rs2106261 and rs2200733 when exposed two copies of risk alleles (RERI=2.87, P<1.00×10-4) or exposed to one additional copy of risk allele (RERI=1.29, P<1.00×10-4). The INTERSNP program identified significant genotypic interaction between rs2106261 and rs2200733 under an additive by additive model (OR=0.85, 95% CI: 0.74-0.97, P=0.02). Mechanistically, PITX2c negatively regulates expression of miR-1, which negatively regulates expression of ZFHX3, resulting in a positive regulation of ZFHX3 by PITX2c; ZFHX3 positively regulates expression of PITX2C, resulting in a cyclic loop of cross-regulation between ZFHX3 and PITX2c. Both ZFHX3 and PITX2c regulate expression of NPPA, TBX5 and NKX2.5. These results suggest that cyclic cross-regulation of gene expression is a molecular basis for gene-gene interactions involved in genetics of complex disease traits. [ABSTRACT FROM AUTHOR]

Hypoxia stimulates angiogenesis under a variety of pathological conditions, including malignant tumors by inducing expression of angiogenic factors such as VEGFA. Surprisingly, here we report significant association between down-regulation of a new angiogenic factor AGGF1 and high-grade urothelial carcinoma. The proportion of strong AGGF1 expression cases was significantly lower in the high-grade urothelial carcinoma group than that in the low-grade urothelial carcinoma group (P =1.40×10−5) or than that in the normal urothelium tissue group (P =2.11×10−4). We hypothesized that tumor hypoxia was responsible for differential expression of the AGGF1 protein in low- and high-grade urothelial carcinomas, and therefore investigated the molecular regulatory mechanism for AGGF1 expression under hypoxia. Under hypoxic conditions, AGGF1 protein levels declined without any change in mRNA levels and protein stability. Hypoxia-induced down-regulation of AGGF1 was mediated by miR-27a. Overexpression of miR-27a suppressed AGGF1 expression through translational inhibition, but not by RNA degradation. Moreover, the hypoxia-induced decrease of AGGF1 expression disappeared after miR-27a expression was inhibited. Furthermore, down-regulation of AGGF1 reduced hypoxia-induced apoptosis in cancer cells. Taken together, the results of this study indicate that (1) hypoxia down-regulates expression of the AGGF1 protein, but not AGGF1 mRNA, by inducing expression of miR-27a; (2) Down-regulation of AGGF1 had an apparent protective role for cancer cells under hypoxia; (3) Down-regulation of the AGGF1 protein confers a significant risk of high-grade human urothelial bladder carcinoma. [ABSTRACT FROM AUTHOR]

The development of the female gametophyte (FG) is one of the key processes of life cycle alteration between the haploid gametophyte and the diploid sporophytes in plants and it is required for successful seed development after fertilization. It is well demonstrated that free nuclear mitosis (FNM) of FG is crucial for the development of the ovule. However, studies of the molecular mechanism of ovule and FG development focused mainly on angiosperms, such as Arabidopsis thaliana and further investigation of gymnosperms remains to be completed. Here, Illumina sequencing of six transcriptomic libraries obtained from developing and abortive ovules at different stages during free nuclear mitosis of magagametophyte (FNMM) was used to acquire transcriptome data and gene expression profiles of Pinus tabulaeformis. Six cDNA libraries generated a total of 71.0 million high-quality clean reads that aligned with 63,449 unigenes and the comparison between developing and abortive ovules identified 7174 differentially expressed genes (DEGs). From the functional annotation results, DEGs involved in the cell cycle and phytohormone regulation were highlighted to reveal their biological importance in ovule development. Furthermore, validation of DEGs from the phytohormone signal transduction pathway was performed using quantitative real-time PCR analysis, revealing the dynamics of transcriptional networks and potential key components in the regulation of FG development in P. tabulaeformis were identified. These findings provide new insights into the regulatory mechanisms of ovule development in woody gymnosperms. [ABSTRACT FROM AUTHOR]